Nitrosative stress under microaerobic conditions triggers inositol metabolism in Pseudomonas extremaustralis

Bacteria are exposed to reactive oxygen and nitrogen species that provoke oxidative and nitrosative stress which can lead to macromolecule damage. Coping with stress conditions involves the adjustment of cellular responses, which helps to address metabolic challenges. In this study, we performed a global transcriptomic analysis of the response of Pseudomonas extremaustralis to nitrosative stress, induced by S-nitrosoglutathione (GSNO), a nitric oxide donor, under microaerobic conditions. The analysis revealed the upregulation of genes associated with inositol catabolism; a compound widely distributed in nature whose metabolism in bacteria has aroused interest. The RNAseq data also showed heightened expression of genes involved in essential cellular processes like transcription, translation, amino acid transport and biosynthesis, as well as in stress resistance including iron-dependent superoxide dismutase, alkyl hydroperoxide reductase, thioredoxin, and glutathione S-transferase in response to GSNO. Furthermore, GSNO exposure differentially affected the expression of genes encoding nitrosylation target proteins, encompassing metalloproteins and proteins with free cysteine and /or tyrosine residues. Notably, genes associated with iron metabolism, such as pyoverdine synthesis and iron transporter genes, showed activation in the presence of GSNO, likely as response to enhanced protein turnover. Physiological assays demonstrated that P. extremaustralis can utilize inositol proficiently under both aerobic and microaerobic conditions, achieving growth comparable to glucose-supplemented cultures. Moreover, supplementing the culture medium with inositol enhances the stress tolerance of P. extremaustralis against combined oxidative-nitrosative stress. Concordant with the heightened expression of pyoverdine genes under nitrosative stress, elevated pyoverdine production was observed when myo-inositol was added to the culture medium. These findings highlight the influence of nitrosative stress on proteins susceptible to nitrosylation and iron metabolism. Furthermore, the activation of myo-inositol catabolism emerges as a protective mechanism against nitrosative stress, shedding light on this pathway in bacterial systems, and holding significance in the adaptation to unfavorable conditions.


Introduction
Bacterial survival and adaptability in changing environments depend on different resistance mechanisms against several stress agents.Free radicals are highly reactive species, mainly originated from energy producing processes like aerobic and anaerobic respiration, causing damage to different macromolecules.Reactive oxygen species (ROS) are produced during aerobic respiration, while anaerobic respiration, utilizing nitrate as electron acceptor, generates reactive nitrogen species (RNS).ROS and RNS trigger oxidative or nitrosative stress, respectively.RNS include nitric oxide (NO) and its derivatives peroxynitrite (ONOO − ), nitrosothiols formed through reaction with thiol groups, and nitrotyrosine from nitration of tyrosine by NO, ONOO − or NO2 - (Crack et al., 2014;Czaja, 2017;Tharmalingam et al., 2017).Nitrosative and oxidative stress can also be induced from various environmental compounds, like pollutants, xenobiotics, and antimicrobials, and from UV radiation (Aranda-Rivera et al., 2022).NO is also produced by the mammalian immune system and acts as a modulator molecule in both mammals and plants (Del Río, 2015;Lelieveld et al., 2021;Li et al., 2020).Nitrosative stress can lead to DNA and macromolecular damage, including protein oxidation.Some residues or structures are more susceptible to the RNS or ROS attack such as iron-sulfur (Fe-S) clusters, metal centers, tyrosine and cysteine residues, and thiols (Zaffagnini et al., 2012).The radical attack to these residues or clusters results in both reversible and irreversible damage, leading to alterations in function and structure (Robinson et al., 2014).
Pseudomonas species exhibit a wide variety of energy-generation metabolisms spanning from aerobic to anaerobic, involving NO3 -reduction, complete denitrification process to N2 and even fermentation of arginine or pyruvate (Arai, 2011;Tribelli et al., 2019).These energy-generating pathways enable Pseudomonas species to thrive in diverse environmental niches, including soil, water, and host-associated habitats, and they play an integral role in the ecological and physiological success of these microorganisms.Pseudomonas extremaustralis is an Antarctic bacterium, capable to growth under different temperatures and oxygen availability.The denitrification process in this bacterium is incomplete, due to the absence of nir genes which encode the enzymes required for the reduction of NO2 -to NO, but interestingly, harbors all the nor and nos genes associated with nitric and nitrous oxide reduction, respectively (Raiger Iustman et al., 2015).
The bacterial adaptation to different stress conditions entails changes leading to solve metabolic and structural challenges, such us alterations or damage in DNA, RNA and proteins and envelopes, among others.Some strategies can involve the utilization of alternative pathways to exploit different carbon sources.Thus, when P. putida was grown at 10°C, the uptake and assimilation of branched-chain amino acids alongside the activation of the 2-methylcitrate pathway to generate succinate and pyruvate has been identified as mechanism employed to cope with stress when central metabolism is downregulated (Fonseca et al., 2011).Likewise, in cold conditions genes involved in primary metabolism were downregulated in P. extremaustralis whereas genes linked to ethanol oxidation were activated, showing that this secondary pathway resulted essential for cold growth (Tribelli et al., 2015).In P. aeruginosa has been reported that under oxidative stress the glyoxylate shunt, an alternative to the tricarboxylic acid cycle, increases bacterial survival allowing the utilization of acetate and fatty acids as carbon sources (da Cruz Nizer et al., 2021).
Considering the importance of RNS derived from both anaerobic respiration and other biotic and abiotic environmental reactions, we evaluated the global transcriptomic response of P. extremaustralis to nitrosative stress under low O2 tensions using Snitrosoglutation (GSNO), as a NO donor compound.Our findings show that exposure to GSNO under microaerobic conditions prompted adjustments in central metabolic pathways including iron metabolism along with the upregulation of genes associated with the inositol catabolism.Notably, myo-inositol arise as a carbon source supporting P. extremaustralis growth and increased nitro-oxidative stress resistance and pyoverdine production.These findings highlight the versatility and adaptability of P. extremaustralis to withstand nitrosative stress, by displaying an alternative metabolism, thus contributing to the understanding of the survival mechanisms employed by microorganisms in extreme environments.

Strains and culture conditions
P. extremaustralis 14-3b (DSM 25547), a species isolated from the Antarctica (López et al., 2009) was used through the experiments.Bacterial cultures were grown in Lysogeny Broth medium (LB) at 30 °C.Microaerobic cultures were incubated in sealed bottles with a 1:2 medium-to-flask volume ratio and 50 rpm agitation.Microaerobic culture's medium was supplemented with 0.8 g/l KNO3.
GSNO effect on P. extremaustralis' survival and growth was determined in microaerobic cultures exposed to 1, 10 and 100 µM of GSNO.Bacterial growth was evaluated by OD600nm measurement after adding the different concentrations of GSNO at T=0. Optical density was monitored over 24 h at 30min intervals using an automated plate reader (BMG OPTIMA FLUOstar).For survival experiments, cultures were grown for 24h and further exposed to the different concentrations of GSNO for 1h.Afterwards, appropriate dilutions of control cultures (m-C) or cultures exposed to GSNO were plated in LB agar and incubated at 30°C.Colony-forming units per ml (CFU / ml) was determined and the survival percentage was calculated with respect to control cultures.

RNA extraction and RNA library preparation
Cultures were microaerobically grown in LB medium for 24 h and then incubated for 1 h with 100 µM S-nitrosoglutatione (GSNO, Sigma Aldrich) (m-NS condition) or with the addition of sterile water (m-C).Total RNA was isolated from 6 ml of P. extremaustralis m-C and m-NS cultures using the Trizol method.Samples were treated with DNAse I and were validated using an Agilent 2100 Bioanalyzer (Agilent Technologies).To improve the quality of the readings, ribosomal RNA was depleted from the samples, using the RiboZERO Kit (Illumina), following the manufacturer's instructions.Libraries were prepared using the TruSeq RNA Library Prep Kit v2 (Illumina).Mass sequencing was performed using NextSeq 550 platform with a single-end protocol.For each condition duplicated independent RNA extraction and libraries were used.

RNA-seq data analysis
Reads were preprocessed using the Trimmomatic tool (Bolger et al., 2014) by eliminating adapters and low-quality sequences.Reads' quality was evaluated using the Fast QC tool (www.bioinformatics.babraham.ac.uk/projects/fastqc/).
Reads alignment and assembly to the P. extremaustralis genome, transcript identification and abundance quantification was carried out using the Rockhopper software (Tjaden, 2020).Reads were normalized per kilobase per million mapped reads (RPKM).Differential gene expression was considered only with P < 0.05 and Q < 0.05.Concordance between the independent replicates for each of the analyzed conditions was verified by performing a Spearman correlation analysis of normalized counts (Fig. S1).

Prediction of oxidation and nitrosylation targets
The Target-Pathogen tool (Sosa et al., 2018) was used to identify proteins with possible oxidation and / or nitrosylation target sites (Free Cys or Tyr residues or metal biding clusters) within the P. extremaustralis genome.

Quantitative Real Time PCR Experiments (RT qPCR)
Total RNA of P. extremaustralis microaerobic and m-NS cultures was isolated using the Total RNA Extraction Kit (RBC Biosciences).After treatment with DNaseI, cDNA was obtained using random hexamers (Promega) and Revert Aid reverse transcriptase (ThermoFisher Scientific) according to the manufacturer's instructions.RT qPCR was performed using a MyiQ2 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, USA) and Real-Time PCR mix (EvaGreen qPCR Mix Plus no ROX).The expression of target genes was evaluated using the following primers: motB forward 5′  (Livak & Schmittgen, 2001) was used to calculate the relative fold gene expression of individual genes.

Bacterial growth using myo-inositol as sole carbon source
Myo-inositol metabolism was evaluated in E2 medium (Lageveen et al., 1988) supplemented with 10 g/l of myo-inositol or glucose under aerobic and microaerobic conditions.Microaerobic cultures were performed as described above while for aerobic experiments, cultures were incubated in Erlenmeyer flasks using 1:10 medium-to-flask volume ratio and 200 rpm agitation.Aerobic and microaerobic cultures were incubated for 24h and 48h, respectively.
Biofilm formation was assayed in E2 medium supplemented with myo-inositol or glucose and KNO3 in polystyrene microtiter plates with an initial OD600nm of 0.025.After 48h of static incubation at 30°C, OD600nm of planktonic cells (Planktonic cells absorbance, PCA) was measured and biofilms were quantified using the standard crystal violet method (O'Toole & Kolter, 1998).Briefly, attached cells were stained with 200 μl of 0.1% crystal violet, further washed and the colorant was solubilized with absolute ethanol.Crystal violet solution was transferred to flat bottom microtiter plates and OD570nm (Crystal violet absorbance CVA) was measured with a BMG OPTIMA FLUOstar microplate reader.
Biofilm formation index was defined as CVA/PCA.

Pyoverdine production
Pyoverdine production of microaerobic cultures was analyzed in iron-limited E2 medium, without microelements, supplemented with myo-inositol or glucose as carbon source and KNO3.After 48h pyoverdines in the culture supernatants were determined by measuring integrated fluorescence emission between 445-460 nm after excitation at 420 nm (Olmo et al., 2003;Ruiz et al., 2015).The values were expressed relative to cell dry weight per ml.

Survival and stress experiments
P. extremaustralis was cultured for 48h under microaerobic conditions in E2 cultures supplemented with myo-inositol or glucose and KNO3 (0.8 g/l).Cultures were exposed to 100 µM GSNO (m-NS) for 1h and cell viability was determined in plate assay.
Growth inhibition in response to oxidative or combined nitro-oxidative stress was evaluated by filter disk assay by growing P. extremaustralis in E2 medium supplemented with glucose or myo-inositol and increasing KNO3 concentrations of 0.8, 1.5 and 2.5 g/l that led to nitrite accumulation.Control cultures without nitrate were performed (oxidative stress only).LB plates were seeded with the different cultures and Whatman n° 1 filter discs (6 mm) were impregnated with 5 μl of 30% (v/v) H2O2 (Merck) as previously described (Ayub et al., 2004).Plates were incubated overnight and the diameter of the halo was determined using the software ImageJ (Schneider et al., 2012).Nitrite concentration in the supernatant of cultures was determined following the method described by (Gerhard et al., 1981) and values were normalized to cell dry weight.

Data availability
RNA-seq data were deposited in the European Molecular Biology Laboratory under accession number E-MTAB-11689.

Statistical analysis
Differences between means were determined through the Student's t test with confidence levels at > 95% in which P < 0.05 was considered as statistically significant.
A Fisher's exact test was performed to analyze the nitrosylation targets proteins.1 or 2ways ANOVA with multiple comparison were used when correspond.

Effects of GSNO exposure in P. extremaustralis
Nitrosative stress effect on P. extremaustralis survival and growth under microaerobic conditions was evaluated using different S-nitrosoglutation (GSNO) concentrations.
Growth in presence of different concentrations of GSNO was similar to the control culture except for 100 µM that showed a decrease at the stationary phase (Fig. S2).Moreover, survival experiments showed that after 1h GSNO exposure, a significant decrease in bacterial viable counts (CFU/ml) was observed comparing with the control culture (Fig. 1).These results indicate GSNO exposure affected P. extremaustralis survival.Thus, we chose 100 µM GSNO (m-NS) exposure for 1h which provoked a 75% drop in survival in microaerobic cultures for RNAseq experiments.).Among them, 86 genes were found to be repressed and 163 over-expressed, representing 1.4% and 2.7% of the total, respectively (Fig. S3a, Table S1a).Several genes with differential expression did not have defined associated functions and were classified as "hypothetical", representing 11.6% and 21% of the over-expressed and repressed genes, respectively.Among genes with a known predicted function, P. extremaustralis under m-NS showed an increased expression of genes involved in carbon, RNA, DNA and iron metabolism (Table S1a).
RNA-seq results were validated by analyzing the expression of three selected genes, motB, flab, and hbo using qRT PCR (Fig. S2b).In concordance with the RNA-seq results, we found higher expression levels of motB, encoding a flagellar motor rotation protein, in m-NS vs. m-C, whereas we observed a decrease in hbo (Fig. S2b, Table S1a) and no differences in flaB expression between conditions.

P. extremaustralis' response to nitrosative stress
To elucidate possible functional relationships, differentially expressed genes were categorized into functional groups (Fig. 2a).GSNO exposure caused increased expression of genes encoding different RNS detoxifying enzymes such as an irondependent superoxide dismutase (PE143B_0125285) and an alkyl hydroperoxide reductase (ahpC), a thioredoxin (PE143B_0111145) and a glutathione S-transferase (PE143B_0109905).A gene encoding another glutathione S-transferase (PE143B_0100180) was repressed in m-NS along with hbO (Fig. 2a, Table S1a).
Transcriptomic analysis showed an increase in mRNA expression of iron and heme transporters coding genes such as ctaB, ybaN, irpA and afnA (Fig 2b, Table S1a).In m-NS we found upregulation in genes related to pyoverdine siderophore biosynthesis, a key function for iron uptake, including pdvA, pdvM, pdvF, fpvA, fpvE and fpvK (Fig. 2b, Table S1a).Moreover, the arginase coding gene, was also upregulated in m-NS comparing with m-C.Arginase catalyzes the conversion of arginine to ornithine, which can then serve as a substrate for pyoverdine biosynthesis (Fig. 2b, Table S1a).
Iron and other metals are key components of iron-sulfur proteins that could be target of RNS, leading to protein inactivation and triggering cellular signaling (Seth et al., 2020).
Therefore, we analyzed other cellular functions related with transcription and protein turn-over.GSNO treated cultures showed an increase in expression of RNA helicase (PE143B_0107010), RNA polymerase associated protein RapA, transcriptional terminator nusB (PE143B_0123570) and 11 ribosomal proteins coding genes (Table S1a).In concordance, a gene encoding translation initiation inhibitor was repressed (Table S1a).To deeply analyze the protein turnover related with RNS attack we performed an analysis using Target Pathogen platform to detect genes encoding proteins with free tyrosine or cysteine as well as metal binding sites which are reactive to RNS (Crack et al., 2014).Among differentially expressed genes in m-NS we found that nitrosylable proteins were over-represented in comparison to non-nitrosylable when total genes of P. extremaustralis were considered (Fisher's exact test, P = 0.0323).Overall, our results suggest that nitrosative stress under microaerobic conditions provokes an increase in the expression of genes involved in transcription and translation processes, probably to compensate the damage to proteins with Fe-S clusters or metal binding (Fig. 2c).

Inositol metabolism in P. extremaustralis
Remarkably, RNAseq results showed increased expression of genes related to inositol catabolism which involves enzymes that convert inositol into acetyl-CoA and dihydroxyacetone phosphate (DHAP) (Fig. 3a, Table S1a).
When we compared the inositol catabolic cluster of P. extremaustralis with P. protegens Pf-5, a well-known plant growth promoter species, and P. syringae pv.syringae B728a, a plant pathogen, we found some differences in this genomic region.Unlike P. protegens Pf-5, the genome of P. extremaustralis did not exhibit the presence of iolL.Conversely, we identified a duplication of iolG within this bacterial species.P. syringae pv.syringae B728a presented iolH, which is absent in both P. protegens and P. extremaustralis.
Similar to P. extremaustralis, P. syringae pv.syringae B728a presented two copies of iolG but separated by iolH (Fig. 3b).The iolA gene in P. extremaustralis is located in a different genomic region like in P. protegens and other analyzed Pseudomonas (Sánchez-Gil et al., 2023).However, in P. syringae pv.syringae B728a is included in the iol catabolic cluster (Fig. 3b).Furthermore, we found that the iol gene cluster of P.
extremaustralis exhibited a high degree of intraspecific conservation showing the same organization as 14-3b in all genomes analyzed, including strains USBA 515, DSM17835 T , 2E-UNGS, CSW01,1906, NQ5.As we detected the presence and expression of iol genes, we first tested P.
extremaustralis capability to grow under aerobic conditions using myo-inositol as sole carbon source.Growth was similar between myo-inositol and glucose supplemented cultures reaching an OD600nm value of 2.02 ± 0.15 and 2.22 ± 0.15, respectively.Under microaerobic conditions P. extremaustralis developed an evident biofilm in myo-inositol supplemented cultures (Fig. 4a).Therefore, OD600nm and cellular dry weight (DW) were determined (Fig. 4b).Similar results were obtained for both carbon sources regardless of the approach employed for growth estimation.Biofilm formation was assayed in E2 medium supplemented with glucose or inositol.In concordance, we also found that with myo-inositol supplemented cultures P. extremaustralis showed a higher biofilm index compared to glucose (Fig. 4c).
Additionally, considering the upregulation of coding genes related with pyoverdine biosynthesis after treatment with GSNO, we investigated siderophores production in P.
extremaustralis using myo-inositol as carbon source.Pyoverdine production was higher for myo-inositol supplemented cultures comparing to glucose (Fig. 4d) Relative Fluorescence Units.Fluorescence units were normalized to the cell dry weight/ml.Error bars represent the standard deviation of the mean.

Myo-inositol impact on stress response
Further, we investigated stress resistance when myo-inositol was used as sole carbon source under low oxygen conditions.To analyze the nitrosative stress response, we performed a survival test using microaerobic cultures in E2 medium supplemented with myo-inositol or glucose exposed to GSNO treatment.We found no differences in survival between those treated with GSNO and control cultures despite of the carbon source (Fig. 5a).
P. extremaustralis is only capable to reduce nitrate to nitrite which accumulates in the extracellular medium.Therefore, the resistance to nitro-oxidative stress derivate from the combination of the nitrite accumulated and H2O2 was analyzed in myo-inositol and glucose supplemented cultures, following the scheme shown in Fig. S4.Nitrite production was detected in cultures supplemented with myo-inositol and glucose as sole carbon source and different nitrate concentrations reaching a maximum of 13.553± 1.624, confirming also the myo-inositol utilization in a respiratory pathway (Fig. 5b).
When P. extremaustralis was grown in myo-inositol or glucose supplemented media without nitrate, the oxidative stress resistance was similar for both carbon sources while the nitro-oxidative stress resistance was higher for cultures grown in myo-inositol in all tested KNO3 concentrations (Fig. 5c).

Discussion
Bacterial species are subjected to different stress conditions including lack of nutrients, changes in temperature or oxygen availability and oxidative and nitrosative stress, among others.Oxidative and nitrosative stress can be produced by the imbalance between the ROS or RNS generation and the incapability to detoxify them (Chautrand et al., 2022) .
In response to these stress conditions, bacteria may activate various response mechanisms, such as the production of chaperons and detoxicant enzymes, the adjustment of gene expression, and the modification of their cell envelope composition.
A common effect during stress is the reprograming of transcription and translation processes including the reduction of ribosome biogenesis, the modification of the translational machinery and the regulation of initiation and elongation of certain proteins (Advani & Ivanov, 2019).Interestingly, gene expression during stress involves not only the transcription processes but also the modification of mRNA stability and in overall with translation provokes a transcriptome stability (Kristoffersen et al., 2012;Vargas-Blanco & Shell, 2020).In this work, we found an up-regulation of the transcription and translation machinery probably to compensate the damage caused by RNS particularly on nitrosylable proteins.The Fe-S clusters, that can be found in three different forms, exhibit high capacity of accepting or donating electrons thus are important for redox response and act as redox sensors like the Fnr regulator in Escherichia coli or Anr in Pseudomonas species (Galimand et al., 1991;Golinelli-Cohen & Bouton, 2017;Vernis et al., 2017).These proteins and those containing metals are involved in several essential cellular functions like respiration, central carbon catabolism and RNA and DNA processes and are a target of RNS damage (Vernis et al., 2017) Particularly, it has been reported that the reaction between nitric oxide (NO) with Fe-S clusters lead to protein degradation and breakdown of the cluster generating dinitrosyl iron complexes (Harrop et al., 2008).Our analysis using Target Pathogen platform showed the enrichment of genes encoding nitrosylable proteins in the differentially expressed ones, particularly in those upregulated.Our hypothesis is that to cope with GSNO derived stress the Fe-S and metal binding proteins need to be replaced.This also could explain the upregulation of Fe-uptake related genes observed in presence of GSNO.Iron is involved in the Fenton reaction in presence of H2O2, derived from endogenous or exogenous sources, that could exacerbate the oxidative damage but paradoxically this metal is also necessary for Fe-S and metal protein biogenesis (Reniere, 2018;Winterbourn, 1995) .This complex scenario additionally requires the involvement of enzymes and antioxidant cycles.In presence of GSNO we found upregulated the coding genes of glutathione S-transferase, superoxide dismutase (SOD), thiol-disulfide isomerase, thioredoxins and alkyl hydroperoxide reductase subunit C-like protein.The alkyl hydroperoxide reductase, important during non-lethal stress, SOD as well as the oxidation of thioredoxins in response to oxidative stress depends on NAD(P)H availability and in overall reduction power is needed for the essential process for survival and to cope with stress (Lemire et al., 2017) .Interestingly, the needed of NADH and/or NADPH production during stress could represent a challenge for bacterial cells.Glycolysis and TCA provides the NADH and energy necessaries for bacterial survival but also creates an oxidative scenario through the respiration chain (Zhang et al., 2019).It has been reported several metabolic strategies to obtain energy and reducing power besides glycolysis such as aminoacid catabolism, ethanol oxidation and in P. fluorescens an entire metabolic reprogramming has been reported in presence of oxidative stress to generate NADPH (Singh et al., 2007).In this previous work the authors showed an increase in the activity and expression of malic enzyme (similar to this work in presence of GSNO), glucose-6phosphate dehydrogenase and NADP+-isocitrate dehydrogenase (Singh et al., 2007).
In addition, we found that in presence of GSNO under microaerobic conditions P.
extremaustralis over-expressed several iol catabolic genes including the transcriptional regulator and the iatA gene.We also demonstrated that this bacterium was effectively capable of using inositol as sole carbon source.Inositol is a sugar alcohol present in all domains of life and presents various structural isomers (Michell, 2008), with myo-inositol being the most abundant in nature.Myo-inositol is found in high quantities in plants, including root exudates, and may also originate from the dephosphorylation of phytate, one of the major phosphorus storage molecules for plants (Weber & Fuchs, 2022).
Although recently inositol metabolism in bacteria has received many interest, its role in stress resistance has not been studied.Remarkably, our findings revealed that inositol protects against nitro-oxidative stress, and increases biofilm and pyoverdine production, which are relevant traits for environmental adaptability.Inositol catabolism leads to the production of glyceraldehyde-3 phosphate, and acetyl-CoA both intermediaries of central energy generation metabolism.In addition, iolG2, which was found overexpressed after GSNO exposure, is related to NAPH generation, necessary for the function of antioxidative defenses.
It is worth noting that previous transcriptome analysis of P. extremaustralis performed under cold conditions or under microaerobic conditions with or without oxidative stress did not show the upregulation of inositol catabolic genes (Solar Venero et al., 2019;Tribelli et al., 2015Tribelli et al., , 2018)), indicating a fine-tuning response to nitrosative stress.
Some RNS also play a crucial role in cell signaling.For instance, in plants, NO mediates for various processes including root hair growth, stomatal closure, programmed cell death and other responses that help them adapt to changing environmental conditions (Zaffagnini et al., 2012) whereas in bacteria, NO plays a role as intermediary in denitrification processes, but also in biofilm formation and quorum sensing (Williams & Boon, 2019).The iol gene cluster was identified in both plant-pathogenic bacteria, and rhizospheric plant growth promoting bacteria forming symbiotic root nodules (Weber & Fuchs, 2022).
Recently, the iol gene cluster in Pseudomonas was identified as an important trait in root colonizers, by increasing swimming motility and siderophore production in response to plant derived inositol (Sánchez-Gil et al., 2023).Increased pyoverdine siderophore production in presence of myo-inositol was also observed in this work.P. extremaustralis possesses plant growth-promotion traits, including the ability to solubilize phosphate and produce indole acetic acid (Ibarra, 2017).Since NO is also a signaling molecule released by plants (Sasaki et al., 2016), we hypothesize that NO released by GSNO mimic the NO production of plants, triggering the differential expression of the inositol metabolic pathway conferring an adaptive advantage.
In conclusion, our findings reveal a multifaceted and finely-tuned response mechanism to nitrosative stress that encompasses transcriptional reprogramming, the upregulation of genes encoding key proteins involved in iron homeostasis, and the activation of inositol catabolism.Notably, experimental results point to inositol metabolism as a novel mechanism to cope with stress.

Figure 2 .
Figure 2. Effect of GSNO on gene expression profile.a. Classification of differentially

Figure 3 .
Figure 3. Metabolic pathway associated with inositol catabolism in Pseudomonas

Figure 4 .
Figure 4. Physiological features of P. extremaustralis using myo-inositol o glucose as carbon source.a. Photograph of microaerobic myo-inositol or glucose supplemented

Figure 5 .
Figure 5. Nitrosative and combined nitro-oxidative stress resistance in P.